NASA Technical Reports Server (NTRS) 20000081744: Altered Cytokine Production By Specific Human Peripheral Blood Cell Subsets Immediately Following Spaceflight

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ALTERED CYTOKINE PRODUCTION BY SPECIFIC HUMAN PERIPHERAL BLOOD
CELL SUBSETS IMMEDIATELY FOLLOWING SPACEFLIGHT
Brian E. Crucian t, Michael L. Cubbage 2and Clarence F. Sams 3
tWyle Laboratories, Cell and Molecular Research Laboratories, Houston, Texas; _Baylor College
of Medicine, Pediatrics-Hematol0gy Oncology Department, Houston, Texas; 3NASA-Johnson
Space Center, Cell and Molecular Research Laboratories, Houston, Texas
Address reprint requests to: Clarence F. Sams, Ph.D., NASA-Johnson Space Center, Life Science
Research Laboratories, Mail Code SD3, Houston, Texas 77058, ph: 218-483-7160: fax [phone]
email: csams @ems.jsc.nasa.go v
ABSTRACT
In this study, we have attempted to combine standard immunological assays with the cellular
resolving power of the flow cytometer to positively identify the specific cell types involved in
spaceflight-induced immune alterations. We have obtained whole blood samples from 27
astronauts collected at three timepoints (L- 10, R+0 and R+3) surrounding four recent space
shuttle missions. The duration of these missions ranged from I0 to 18 days. Assays performed
included serum/urine cortisol, comprehensive subset phenotyping, assessment of cellular
activation markers and intracellular cytokine production following mitogenic stimulation.
Absolute levels of peripheral granuloeytes were significantly elevated following spaceflight, but
the levels of circulating lymphocytes and monoCytes were unchanged. Lymphocyte subset analysis
demonstrated trends towards a decreased percentage ofT cells and an increased percentage of B
cells. Nearly all of the astronauts exhibited an increased CD4:CD8 ratio, which was dramatic in
some individuals. Assessment of memory (CD45RA+) vs. naive (CD45RO+) CD4+ T cell
subsets was more ambiguous, with subjects tending to group more as a flight crew. All subjects
from one mission demonstrated an increased CD45RA:CD45RO ratio, while all subjects from
another mission demonstrated a decreased ratio. While no significant trend was seen in the
monocyte population as defined by scatter, a decreased percentage of the CDI4+ CD 16+
monocyte subset was seen following spaceflight in all subjects tested. In general, most of the
cellular changes described above which were assessed at R+0 and compared to L-10 trended to
pre-flight levels by R+3. Although no significant differences were seen in the expression of the
cellular activation markers CD69 and CD25 following exposure to microgravity, significant
alterations were seen in cytokine production in response to mitogenic activation for specific
subsets. T cell (CD3+) production of IL-2 was significantly decreased after at R+0 as was IL-2
production by both CD4+ and CD8+ T cell subsets for most subjects. Production of IF'N'/did not
appear to be affected by microgravity exposure in either T cells in general or in the CD8+ T cell
subset. There was a spaceflight-induced decrease in IFN_, production in the CD4+ T cell subset,
however it did not reach statistical significance. Serum and urine stress-hormone analysis
indicated significant physiologic stresses in astronauts following spaceflight. In summary, these
results demonstrate alterations in the peripheral immune system of astronauts immediately after
spaceflight of 10 to 18 days duration and support continued research regarding microgravity and
immunology (including in-flight sampling) prior to routine long-term spaceflight for astronauts.
INTRODUCTION
Dysregulation of the immune system has been a well-documented effect of human exposure to
a microgravity environment during spaceflight (1). Extensive ground based studies using
clinorotation cell culture, animal restraint unloading and human long term bed-rest have
demonstrated a variety of the effects of simulated microgravity on the immune system. These
effects have included altered cytokine production (2,3,4), reduced proliferative/activation
responses (5,6) and altered signal transduction pathways (5,7). In addition to these ground based
models, studies performed on subjects exposed to the space environment during and immediately
after landing have also demonstrated altered immune responses (8). These data have included
altered cytokine production (9,10,11) and altered distribution of peripheral immune cells (12,13).
Recent reports have documented immunological studies performed in-flight (14). When tested
during spaceflight, delayed type hypersensitivity was reduced, indicating a dysregulation of cell-
mediated immune function (15,16). The mechanisms which produce this dysregulation have not
been clearly established. In addition to the direct effects of microgravity on cellular physiology
other factors may also have influence. These may include radiation, changes in calcium usage in
the musculosystem and psychological issues such as stress.
The production of cytokines plays a critical role in the ability of a host to mount an immune
response against an invading microorganism. In addition, the particular cells producing cytokines
and the specific cytokines produces directly determine the type and magnitude and duration of the
immune response. It naturally follows that a decreased ability to produce cytokines can result in
potentially significant immune alteration and a reduced ability to fight infections.
In the 1990s the ability to detect cytokine production in individual positively identified cells by
flow cytometry was developed (17,18,19). Conventional assays to detect cytokine production
have several drawbacks. Bioassays are not necessari/y cytokine-specific in that they measure
functional properties. Supernatant cytokine protein can be readily measured by ELISA methods,
but unless a highly purified cell population was cultured this method does not identify the
population of cells responsible for the cytokine production (i.e.: PBMC's). In addition, the results
of ELISA assays reflect the net outcome of produced, absorbed and degraded cytokine and do
not necessarily distinguish between biologically active and inactive substances. In situ
hybridization and RT-PCR detect cytokine gene expression and are highly sensitive but they are
labor-intensive and the presence of cytokine RNA does not guarantee translation to cytokine
protein. The advantages of this flow cytometric detection ofcytokines are the detection of
cytokine protein, as opposed to cytokine RNA and the ability to examine the production of
cytokines by individual cell populations while still culturing mixed populations of cells (PBMCs)
to retain the various cell-cell interactions. The assessment of cytokine production has been shown
to have great value in the research laboratory for investigating basic biological interactions
(20,21,22). Recently, flow eytometric cytokine detection has been demonstrated to have
diagnostic value in clinical situations (23,24) and will conceivably enter into clinical use for the
rapid and routine monitoring of cytokine production and peripheral immune balance.
In this study, we have attempted to combine standard immunological assays with the cellular
resolving power of the flow cytometer to positively identify the specific cell types involved in
microgravity induced immune dysregulation. Blood was collected from 27 astronauts
(representing four recent space shuttle missions) both before launch and at 2 points after landing.
A comprehensive battery of standard immunological assays was performed, in addition to the
detection of cytokine responses by flow cytometry. Due to the limitations in available sampling
foreachflightallassayswerenotperformedonallshuttleflights,howeverallassayswere
performedonsamplesfromatleasttwo ofthefourshuttle flights (intracellular cytokine
assessment was performed on three flights). We found that the ability to produce IL-2 was
depressed after spaceflight, and that CD4+ and CD8+ T cell subsets seemed equally sensitive to
the effect. Regarding IFN_, production, only the CD4+ T cell subset demonstrated decreased
production after spaceflight. IFN'y production by the CD8+ subset appeared largely unchanged.
MATERIALS AND METHODS
Whole blood collection. Whole blood was collected from 27 healthy astronaut donors at three
timepoints surrounding space shuttle flights. These subject"s consist of the entire crew from four
separate shuttle missions, which varied in length from 10 to 18 days. Blood collection was
obtained 10 days prior to launch (L- 10), immediately after landing (R+0) and three days after
landing (L+3). The whole blood was collected into EDTA Vacutainer tubes (Becton Dickinson,
Mountain View, CA). [nformed consent was obtained from all participants.
Hematology analysis. Standard clinical hematology analysis was performed according to the
manufacturers instructions on a Coulter MaxM hematology analyzer. Results obtained included a
standard complete blood count and WBC differential, including cell percentages and absolute
counts.
Cell culture for activation. Four ml of whole anticoagulated blood was diluted I: 1 with sterile
phosphate buffered saline (PBS) and subjected to centrifugation over a Ficoll-Hypaque density
gradient (Pharmacia, Upsala Sweden) to isolate PBMC's. The isolated cells were washed 3 times
in sterile PBS and resuspended in RPMI medium 1640 (Gibco BRL, Grand Island, NY)
containing 10% fetal bovine serum (GIBCO) and 1 x 105 mg/ml penicillin and streptomycin
(GIBCO). The final concentration of cells was determined using a hemacytometer. A final
concentration ranging from 0.7-1.3 x lO6cells/ml was used in all experiments. For intraceUular
cytokine analysis cells were cultured in either medium containing 10 ng/ml PMA and 1mg/ml
ionomycin for 5 hours or medium containing 10 ug/ml with 5 I.tg/ml lipopolysaccharide E.coli
strain 011:B4 (LPS; Sigma). In all cases 3 mM monensin (Sigma) was added to the PBMC
cultures to shut down extraeellular transport of cytokines and allow intraceUular accumulation.
For assessment of the cell surface activation markers CD69 and CD25 in response to stimulus
isolated PBMC's were cultured in medium containing 10 _ghrd PHA for 24 hours.
Surface marker staining. For WBC surface marker analysis 100 _! of peripheral whole blood
was stained along with the manufactures suggested amount of FLl:fluorescein isothiocyanate
(FITC), FL2:phycoerythrin (PE) or FL3:Peridinin Chlorophyll Protein (PercP) labeled monoclonal
antibodies in 100 l.tl of phosphate buffered saline (PBS). The mixtures were incubated on ice for
15 minutes, 3 ml of.85% ammonium chloride was added and the mixture was incubated in the
dark for 5 minutes to lyse the RBC's, washed 3 times with 3 ml of PBS and resuspended in 0.5 ml
PBS with 1% paraformaldehyde.
For the assessment of CD69 and CD25 the cells (activated in culture) were washed and
•resuspended in 5001.tl PBS to which the manufacturers suggested amount of labeled monoclonal
antibodies to CD69 and CD25 were added. Following incubation for 10 minutes the cells were
washed and resuspended in 5001.tl of PBS containing 1.0% paraforrnaldehyde prior to analysis.
Intracellular cytokine staining. An antibody configuration was selected which assessed T cell
production of [FNy and [L-2 in T cells (including production from CD4+ and CD8+ T cell
subsets) in response to PBMC stimulation with PMA and ionomycin. Surface markers were
stained f'u'stby resuspending the cells in 200 ml of PBS to which 1.0 mg of PerCP labeled mouse
monoclonal antibody to CD3, CD4 or CD8 was added, samples were incubated at R.T. for 10
minutes and then washed with PBS. The PBMC's were then fixed in 200 ml of 4.0%
paraformaldehyde in PBS for l0 minutes, then washed again in PBS. To detect intracellular
production of IFN_, or IL-2 in conjunction with a surface marker, fixed PBMC's were resuspended
in 200 ml of permeabilization buffer (PB), consisting of 5.0% non-fat dry milk and 0.5% saponin
in PBS to which 0.5 mg of labeled mouse antibody to either IFN 7 or IL-2 was added. Cells were
incubated at room temperature for 25 minutes. The cells were then washed in PBS containing
saponin (PBSS) and resuspended in 1.0% paraformaldehyde for analysis.
To detect intracellular production of [L-ls in conjunction with surface CD 14 in monocytes,
the activated PBMC's were resuspended in 200 ml of PBS to which 1.0 mg of FITC labeled
mouse monoclonal antibody to CDI4 was added and incubated at R.T. for 10 minutes. The
PBMC's were then washed in PBS and fixed in200 ml of 4.0% paraformaldehyde in PBS for 10
minutes, then washed again in PBS. To detect intracellular IL-1o_, the fixed PBMC's were
resuspended in 200 ml of PB with 0.5 mg ofa PE labeled antibody to IL-Io_ (Pharmingen),
incubated for 25 minutes at R.T. and washed with PBSS. The cells were then resuspended in
1.0% paraformaldehyde for analysis.
Flow eytometry analysis. For analysis, a Coulter XL flow cytometer was configured with
appropriate electronic compensation for spectral overlap of the light emitted by the different
fluorochromes. The lymphocyte cluster was identified by plotting forward versus side (90 °) light
scatter. The lymphoid cells were then 'gated' for immunofluorescence analysis, including only the
lymphoid cells in the data analysis. Thresholds for positive were set using isotype matched
control antibodies. Sample analysis was performed using System 2 software on a Coulter-XL flow
cytometer
Serum and urine stress hormone measurement. Analysis of serum and 24-hour urine were
performed using commercially available kits according to the manufacturer's instructions.
Serum/urine cortisol were performed by RIA (DiaSorin, Stillwater, MN); urine aldosterone was
performed by RIA (Diagnostic Product Corporation, Los Angeles, CA); urine ADH was
perfomed by RIA (Diagnostic Systems Laboratories, Inc., Webster, TX); urine
epinephrineJnorepinephrine were performed by RIA (ALPCO, Windham, NH).
Statistical analysis. To test the significance of differences observed as a result of exposure to
microgravity either a one way repeated measures analysis of variance (three groups) or a paired T
test (two groups) was performed. The results obtained from preflight (L-10) testing were
compared to the results obtained from landing day (R+0) testing. Statistical significance was
def'med by P<0.05 and differences which achieved significance are indicated as such in the figures.
RESULTS
White Blood Cell Differential. The data were consistent with significant alterations in the
status of the peripheral immune system upon landing. An analysis of relative percentages of
granulocytes, lymphocytes and monocytes from all four shuttle flights indicated a statistically
significant increase in the percentage of peripheral blood granulocytes, which was accompanicd by
a corresponding significant decrease in the relative percentage of peripheral blood lymphocytcs
(data not shown). Considering that the perccntages of WBC subsets are relative with respect to
each othcr, an analysis of absolute pcripberal blood cell counts was performed. Absolute levels of
peripheral granulocytes were significantly elevated following spaceflight, but the levels of
circulating lymphocytes and monocytes were unchanged (Fig. 1). These data indicate that the
differences in circulating granulocytes had artificially skewed thE'lymphocyte percentage data.
After three days of exposure to unit gravity, the percentages in most of the subjects had returned
to near baseline. No significant alterations regarding levels of circulating monocytes were seen
following spaceflight (Fig. 1).
Surface Marker Subset Analysis, Lymphocyte subset analysis demonstrated a significantly
decreased percentage ofT cells (CD3+) in 10 of 13 subjects (P<0.05, Fig. 2a) and .aslightly
increased percentage of B cells (CD19+) in 9 of 13 subjects (Fig. 2b). No real trend was seen in
the percentage of NK cells (CD16/56+; Fig. 2c). Nearly all (23 of 27) astronauts had an increased
CD4:CD8 ratio, which was dramatic in some individuals (P<0.05, Fig. 2d). For the majority of
the subjects with an increased CD4:CD8 ratio at R+I, the ratios returned to near baseline (L-10)
values within three days after landing (R+3). Assessment of memory (CD45RA+) vs. naive
(CD45RO+) CD4+ T cell subsets was more ambiguous, with subjects tending to group as a flight
crew. All subjects from one mission (7 of 7) demonstrated an increased CD45RA:CD45RO ratio,
while all subjects from another mission (5 orS) demonstrated a decreased ratio (Fig. 2e).
Subjects from a third mission cxhibiteded no significant trend. The reason for this grouping is
unclear. While no significant trend was seen in the monocyte population as defined by scatter, a
significant decrease in the percentage ofCDl4+ CDI6+ monocyte subset was seen in all subjects
tested (P<0.05, Fig. 20. All of the cellular changes described above which were assessed at R+0
trended to pre-flight levels by R+3.
Expression of Cellular Activation Markers. Isolated PBMC's from astronaut whole blood
samples were cultured in thc presence of 10 _tg/ml PHA for 24 hours to assess the capacity of the
cells to respond to a mitogcnic signal. Under normal conditions, the expression of CD69 occurs
early during lymphocyte activation, and the expression of CD25 occurs somewhat later during the
activation process. No significant differences were seen in the expression of the cellular activation
markers CD69 and CD25 following activation in culture for 24 hours at either R+0 or R+3 as
compared to L-10 preflight samples (data not shown).
Intracellular Cytokine Detection by Flow Cytometry, Significant alterations in cytokine
production by specific lymphocyte subsets in response to mitogenic activation were detected by
flow cytometry. Representative flow cytometry scatter plots demonstrating decreased production
of IFNT and IL-2 after spaceflight (R+0), as compared to preflight (L-10) and a three day
recovery after flight (R+3) for one astronaut are presented in figure 3. The mean percentages of
cytol(ine producing cells for all timepoints were determined and are presented in figure 4. In the
grouped subjects, the data indicate a suppression in the ability of stimulated T cells (CD3+) to
produce IL-2 after spaceflight. T cell production of IFNy was not affected. When cytokine
production by the individual T cell subsets (CD4+ and CD8+) was examined,
the suppression of IL-2 production was evident in both the CD4 and the CD8 T cell subsets
(figure 4). In contrast, IFN'y production was reduced in the CD4+ T cell subset while CD8+ T
cell production of IF-'Nyappeared to be unchanged.
To examine the subjects individually, the eytokine production data for the astronauts at L- 10
was normalized to 100% (Fig. 5). Thus, the relative increases or decreases at R+0 may be
visualized for all subjects while maintaining the ability to look at individual data points. T cell
(CD3+) production of IL-2 was decreased after at R+0 in all subjects assayed (7 of 7; Fig. 5a).
In these same subjects, however, no trend emerged regarding the T cell expression of IFNT(Fig.
5d). Specific T cell subset (CD4 vs. CD8) cytokine production was assessed for a larger
population of astronauts. It was found that the ability to produce IL-2 in response to mitogenic
stimulus was depressed for both the CD4+ subset (11 of 12 subjects; Fig 5b) and for the CD8+
subset (18 of 20 subjects; Fig 5c).
Generalized T cell (CD3+) production of IFN 7 was altered, although this assay was performed
on only one mission with a sample size of seven. The production of WN'f by specific T cell
subsets was investigated on a larger sample size spanning three missions. When T cell production
of IFN 7 was assessed in the context of CD4+ and CDS+ subsets, the CD4+ T cell subset appeared
to be more sensitive to the effects of spaceflight. The majority of astronauts tested demonstrated a
decrease in production (10 of 12; p<0.05; Fig. 5e) of IFN_,. The production of IFN_, by CD8+ T
cells was effected by spaceflight as no clear trend emerged (Fig. 5f).
The ability of monocytes to produce IL-lct in response to mitogenic stimulus was also
assessed. No dramatic alterations and no clear trends emerged for the majority of the subjects (14
of 20; data not shown), however for a small number of subjects (6 of 20) a dramatic increase in
IL-1 o_production was seen at R+0. For these subjects these responses trended towards L-I0
levels by R+3. This population represented the entire crew for one space shuttle flight out of the
three flights for which this assay was performed. A comparison of the mean percentage of IL-10t
producing monocytes for L-10 and R+0 indicated the increase in production was statistically
significant (p<0.05).
Serum and urine chemistries. Serum and urine stress hormone analysis was performed on
subjects both pre-flight and immediately post-flight to investigate the physiologic stress resulting
from spaceflight/landing. Serum cortisol and ACTH, as well as urine cortisol, aldosterone, ADH,
epinipherine and norepinepherine were assessed. All mean measurements were elevated post-
flight, as compared to preflight values (Fig. 6). The elevations for urine cortisol, aldosterone and
norepinepherine achieved statistical significance (P<0.05, Fig .6). These results indicate that the
astronaut subjects were subjected to significant physical stresses during, or immediately following
spaceflight.
DISCUSSION
The dysregulation of the immune system is a well-documented result of prolonged exposure to
microgravity. Several studies have identified altered cytokine production profiles in subjects
exposed to microgravity for prolonged periods (9,10,11). In addition, ground based studies have
also indicated that simulated microgravity also results in a depressed immune response and
reduced capacity to produce specific cytokines (2,3,4,5,6,7).
Here we confLrm the findings of others regarding alterations in the peripheral blood immune
cell subsets. In particular, we demonstrate an increase in the percentage of peripheral blood
granulocytes and a decrease in percentages of lymphocytes immediately following spaceflight.
This is consistent with the results seen by Stowe, et al who recently reported on neutrophil
percentages and function following spaceflight (25). Since the _"rrcentages of WBC subsets are
relative with respect to each other, an analysis of absolute peripheral blood cell counts was
performed. Absolute levels of peripheral granulocytes were significantly elevated following
spaceflight and the absolute levels of lymphocytes and monocytes were unchanged (Fig. 1).
These data indicate that the differences in circulating granulocytes had artificially skewed the
lymphocyte percentage data. Regarding specific lymphocyte subsets, we report here that the T
cell CD4:CD8 subset ratio increased following spaceflight. It is likely that this result would be
attributed to decreased levels of CD8+ T cells, as opposed to increased levels of CD4+ T ceils,
given the overall decrease in the levels of total T cells. These results will require confirmation in
the future on a larger sample size and perhaps using recently developed methods to directly
quantify absolute counts during flow cytometry analysis. No significant trends emerged regarding
levels of B ceils and NK ceils following spaceflight in our study. In addition, ratios of memory
(CD45RO+) to naive (CD45RA+) T cells did not demonstrate significant trends in our study.
This was not unexpected, as the duration of these flights was probably not long enough to result
in a broad expansion of memory cells, and since no significant lengthy illnesses were reported
during these four shuttle flights. No alterations in monocyte levels were detected when analyzing
by scatter properties alone. The recently described subset of CD 14+/CD 16+ peripheral blood
monocytes was also assessed in this study. This unique subset, functionally appearing like tissue
macrophages, has recently been identified and associated with specific disease processes (26,27).
This subset comprises approximately 10% of the total peripheral blood monocyte population.
Our preflight CD 14+ CD 16+ monocyte values correlated well with that value, and a significant
decrease was observed postflight. Values rebounded to levels at or above the preflight value by
R+3. Although the clinical significance of this finding is unknown, this subset has been reported
to be proinflammatory in nature. Levels were reported to be decrease in subjects after extreme
exercise (26). This decrease may be an acute response to the physical stresses of landing. In-
flight measures will be required to further investigate the role of these cells in spaceflight immune
responses.
In this study we have identified specific subpopulations of immune ceils which demonstrate
alterations in their ability to produce cytokines. This was accomplished by utilizing flow
cytometry, with which cytokines and surface markers can be stained simultaneously, resulting in
positive identification of cytokine producing cells. This technology was used to positively identify
the particular subsets of T cells susceptible to spaceflight-induced cytokine (IFN T and [L-2)
dysregulation. We report here that the ability ofT cells to produce IL-2 was depressed in the
total T cell population, as well as in both the CD4+ subset and the CD8+ subset. This alteration
was statistically significant for all three populations, and results trended towards preflight values
in the R+3 specimens. This indicates that a recovery in IL-2 responsiveness was occurring several
daysafterlanding.An interesting differentiation in the effects of spaceflight was observed for the
T cell subsets regarding the production of IFNy. While total T cell production of [FNy was not
effected in a significant fashion (although tested in a notably small sample size), the CD4+ subset
did demonstrate a significant reduction in IFNy production. This effect was not observed in the
CD8+ subset. Although the individualized data plot for CD4+/IFNy production reveals 5 of 20
astronauts with increased production after spaceflight, statistical analysis of the entire population
demonstrated a significant reduction overall (with 15 of 20 astronauts demonstrating a reduction
for this subset). In the context of the increased CD4:CD8 ratio demonstrated earlier, it is possible
that the bias of the peripheral blood CD4+ T cell population is being shifted away from IFNy
production. Future studies investigating this phenomenon should include a comprehensive study
the effect of microgravity on the Th l/Th2 cytokine balance. Further testing with increased sample
size is clearly indicated to confirm these results. This data suggest that the effects of spaceflight
on cytokine production may differ for both the individual cytokines and also for different cell
subsets responsible for cytokine production.
It should be noted that assessments of biological activity, such as the ones conducted in this
paper, on astronauts returning from spaceflight can be skewed by the psychological and
physiological stresses of landing and readaptation to unit gravity. The serum and urine stress
hormone results reported here indicate that subjects returning from spaceflight have been exposed
to significant physical stress. The only way to overcome this potential shortcoming is to perform
testing during spaceflight. Unfortunately, the technical limitation of experimentation in spaceflight
has precluded performing many sophisticated laboratory analyses in the space environment. The
development of newer innovative biological spaceflight-compatible hardware for performing
routine immunologic monitoring may overcome some of these problems (28, 29).
As increased numbers of humans will be subjected to spaceflight of increasing duration, the
dysregulation of the human immune system in microgravity is becoming a great concern to the
spaceflight medical community. This is especially true as NASA and other space agencies prepare
for lengthy missions to Mars. Thorough investigation of the effects of spaceflight on the human
immune system may result in the development of effective countermeasures either via
pharmacology or through manipulation of the spaceflight environment (i.e. artificial gravity, or
other mechanisms). It is expected that the International Space Station will serve as an excellent
platform to further extend research into the immunology effects on human physiology, as well as
other related biological sciences. This data would then enhance our ability to develop required
countermeasures and ensure the health and safety of future long-duration spaceflight crew
members.
ACKNOWLEDGMENTS
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